There is a rapidly growing interest in the detection of specific nucleic acid sequences. This interest has not only arisen from the recently disclosed draft nucleotide sequence of the human genome and the presence therein, as well as in the genomes of many other organisms, of an abundant amount of single nucleotide polymorphisms (SNP), but also from marker technologies such as AFLP and the general recognition of the relevance of the detection of specific nucleic acid sequences as an indication of for instance genetically inheritable diseases. The detection of the various alleles of the breast cancer gene BRCA 1 to screen for susceptibility for breast cancer is just one of numerous examples. The recognition that the presence of single nucleotide substitutions (and other types of genetic polymorphisms such as small insertion/deletions; indels) in genes provide a wide variety of information has also attributed to this increased interest. It is now generally recognised that these single nucleotide substitutions are one of the main causes of a significant number of monogenically and multigenically inherited diseases, for instance in humans, or are otherwise involved in the development of complex phenotypes such as performance traits in plants and livestock species. Thus, single nucleotide substitutions are in many cases also related to or at least indicative of important traits in humans, plants and animal species.
Analysis of these single nucleotide substitutions and indels will result in a wealth of valuable information, which will have widespread implications on medicine and agriculture in the widest possible terms. It is for instance generally envisaged that these developments will result in patient-specific medication. To analyse these genetic polymorphisms, there is a growing need for adequate, reliable and fast methods that enable the handling of large numbers of samples and large numbers of (predominantly) SNPs in a high throughput fashion, without significantly compromising the quality of the data obtained. One of the principal methods used for the analysis of the nucleic acids of a known sequence is based on annealing two probes to a target sequence and, when the probes are hybridised adjacently to the target sequence, ligating the probes. This concept is commonly indicated as the Oligonucleotide Ligation Assay or Oligonucleotide Ligation Amplification (OLA)
The OLA-principle has been described, amongst others, in U.S. Pat. No. 4,988,617 (Landegren et al.). This publication discloses a method for determining the nucleic acid sequence in a region of a known nucleic acid sequence having a known possible mutation. To detect the mutation, oligonucleotides are selected to anneal to immediately adjacent segments of the sequence to be determined. One of the selected oligonucleotide probes has an end region wherein one of the end region nucleotides is complementary to either the normal or to the mutated nucleotide at the corresponding position in the known nucleic acid sequence. A ligase is provided which covalently connects the two probes when they are correctly base-paired and are located immediately adjacent to each other. The presence, absence or amount of the linked probes is an indication of the presence absence or amount of the known sequence and/or mutation.
Abbot et al. in WO 96/15271 developed a method for a multiplex ligation amplification procedure comprising the hybridisation and ligation of adjacent probes. These probes are provided with an additional length segment, the sequence of which, according to Abbot et al., is unimportant. The deliberate introduction of length differences intends to facilitate the discrimination on the basis of fragment length in gel-based techniques.
WO 97/45559, WO97/31256, WO98/03673, WO00/56929, WO00/56927, WO00/40755 (Barany et al.) describe methods for the detection of nucleic acid sequence differences by using combinations of ligase detection reactions (LDR) and polymerase chain reactions (PCR). Disclosed are methods comprising annealing allele-specific probe pairs to a target sequence and subsequent ligation with a thermostable ligase. Amplification of the ligated products with fluorescently labelled primers results in a fluorescently labelled amplified product. Detection of the products is based on separation by size or electrophoretic mobility or on an addressable array.
Other variants of OLA-based techniques have been disclosed inter alia in Nilsson et al. Human mutation, 2002, 19, 410-415; Science 1994, 265: 2085-2088; U.S. Pat. No. 5,876,924; WO 98/04745; WO 98/04746; U.S. Pat. No. 6,221,603; WO 03/054511, U.S. Pat. No. 5,521,065, U.S. Pat. No. 5,962,223, EP185494, EP246864, U.S. Pat. No. 6,027,889, EP745140, EP964704, US20030119004, US2003190646, EP1313880.
Recent publications (Hardenbol et al., Nat. Biotechnology 2003, 21, 673-678; Banér et al., Nucleic Acids Research, 2003, 31, e103) have shown that the OLA principle can be highly multiplexed, making it an attractive technique for high throughput SNP genotyping, especially in combination with sequence-based detection platforms, such as the ones used by the authors of these papers. However, in combination with length-based detection platforms, the high multiplex capacity of the OLA technique is difficult to exploit, due to the limited size distribution of the amplification products obtained from ligated probes that can be detectably separated using current (capillary) sequencing instruments when using ligation probes synthesised by chemical means. This is because the upper limit of currently available chemical oligonucleotide synthesis techniques lies at around 100 to 150 basepairs, which is much less than the size range covered by most (capillary) sequencing instruments. Nevertheless, slab-gels or sequencing instruments are powerful detection platforms due to their ease of use, limited hands-on time and relatively low operating costs compared to most commercially available chip (hybridisation) platforms.
Schouten et al. Nucleic Acids Research, 2003, 30, e57; and EP130113 and WO01/61033) have partially countered this limitation of length-based detection due to the length limitation of chemically synthesised ligation probes by preparing the probes using single stranded phage M13. This ensures high quality probes with a uniform length, capable of spanning the entire length window of a (capillary) sequencing instrument or slab gel system for the detection of amplified ligation probes. However, the probe preparation method of Schouten et al. is cumbersome, time-consuming, difficult to automate and therefore costly and not well suited for applications involving many different target sequences. Hence others solutions are still needed to make efficient use of size-based detection platforms for detection of amplified ligation probes.
Van Eijk et al. (WO03/52140; WO03/52141 WO03/52142, Nucleic acids research, 2004, 32(4), e47) have provided a solution to this problem by selectively amplifying subsets of ligated probes using selective AFLP primers such as those described by Vos et al. for AFLP fingerprinting (Vos et al., Nucl. Acids Res., 1995, 21, 4407-4414; EP534858, U.S. Pat. No. 6,045,994, WO93/06239). Although this approach allows selection of particular subsets of ligated probes for co-amplification in the same reaction with a single primer pair, the composition of the amplifiable subsets is fixed and determined by incorporation of the appropriate binding sites for the AFLP primers in the ligation probes when designing them.
With an increasing demand for high throughput multiplex assays, (i.e. assays that are able to address (detect) a large number of target sequences in one sample and that are able to address many samples in a short period of time), one of the less advantageous aspects of many of the probes that are used in the current oligonucleotide ligation assays is the tendency for probe lengths and the length of the corresponding ligation products to increase.
The current methods are able to provide oligonucleotides through nucleotide coupling reactions with a yield of 98.5% per nucleotide. This means that with an increasing length, for each nucleotide in the probe, the yield of the desired full length probe is lowered and the amount of undesired probes (incomplete synthesis products) increases. As a result, to provide for probes of sufficient length and/or sufficient purity, additional steps are needed to purify the probes prior to use in any assay or alternative methods of synthesis are required.
The increasing length of the products of the ligation of probes presents also a disadvantage, in particular with detection systems based on length, but also in case of mass-based detection or hybridisation based detection due to the increasing possibility of cross-hybridisation.
The present inventors have made it their aim to investigate the oligonucleotide ligation assays and to provide assays that can provide the same amount of information of the same quality, only with probes and/or ligation products of shorter and/or more flexible length. It is one of our aims to modify the assay in which these probes are used and to introduce more flexibility.